Methods for calcium phosphate transfection

ABSTRACT

A method is provided for calcium phosphate transfection of a eukaryotic host cell wherein particles comprising calcium phosphate and a desired nucleic acid are grown to an optimal size and then contacted with the host cell under conditions providing a substantially slower particle growth rate, thereby increasing the host cell&#39;s exposure to optimally-sized particles.

This is a continuation of application Ser. No. 08/303,245 filed on Sep.8, 1994, now U.S. Pat. No. 5,484,720, the entire disclosure of which isspecifically incorporated herein by reference and to which applicationpriority is claimed under 35 U.S.C. §120.

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

This invention relates to the field of nucleic acid transfection, endmore particularly to methods for nucleic acid transfection of eukaryoticcells by calcium phosphate co-precipitation.

2. DESCRIPTION OF THE BACKGROUND AND RELATED ART

The ability to introduce foreign DNA into eukaryotic host cells is oneof the principle tools of recombinant DNA technology. Methods fortransfecting eukaryotic host cells with foreign DNA can be broadlygrouped into four categories: (1) direct introduction of cloned DNA bymicroinjection or microparticle bombardment; (2) use of viral vectors;(3) encapsulation within a carrier system; and (4) use of facilitatorssuch as calcium phosphate and diethylaminoethyl (DEAE)-dextran. Of thereagents used as facilitators of DNA transfection, calcium phosphateremains the most widely used because of its simplicity end generaleffectiveness for a wide variety of cell types.

The original protocol for calcium phosphate transfection was describedby Graham and van der Eb, Virology, 52: 456-467 (1973). This method wasmodified by Wigler et el., Proc. Natl. Acad. Sci., 76: 1373-1376 (1979)and by Chen and Okayams, Mol. Cell. Biol., 7: 2745-2752 (1987).Nevertheless, the original and modified protocols yield relatively lowtransfection efficiencies and expression in experiments geared towardstransient or stable DNA transfer. Accordingly, there is still a need foran improved method of calcium phosphate transfection.

This and other objects will be apparent to those skilled in the art.

SUMMARY OF THE INVENTION

The invention provides for a method for introducing a desired nucleicacid into a eukaryotic host cell comprising admixing Ca²⁺, PO₄ ³⁻ andthe desired nucleic acid to form a precipitation mixture; incubating theprecipitation mixture to form particles comprising calcium phosphate andthe desired nucleic acid, and allowing the particles grow to an averagelength of up to about 300 nanometers (nm); performing a step selectedfrom the group consisting of: (1) diluting the precipitation mixture andsimultaneously admixing the precipitation mixture to a eukaryotic hostcell lacking a cell wall to form a transfection mixture with an initialCa²⁺ concentration that is at least ten fold lower than the initial Ca²⁺concentration of the precipitation mixture and wherein the particles arecapable of further growth in the transfection mixture, and (2) dilutingthe precipitation mixture to form a diluted precipitation mixture, andthereafter admixing the diluted precipitation mixture to a eukaryotichost cell lacking a cell wall to form a transfection mixture with aninitial Ca²⁺ concentration that is at least ten fold lower than theinitial Ca²⁺ concentration of the precipitation mixture and wherein theparticles are capable of further growth in the transfection mixture; andincubating the transfection mixture to allow the eukaryotic host cell totake up the particles to form a transfected cell.

The invention further provides a method for introducing a desirednucleic acid into a eukaryotic host cell comprising admixing Ca²⁺, PO₄³⁻ and the desired nucleic acid to form a precipitation mixture;incubating the precipitation mixture for a period of up to about 60seconds to form a precipitate comprising calcium phosphate and thedesired nucleic acid; performing a step selected from the groupconsisting of: (1) diluting the precipitation mixture and simultaneouslyadmixing the precipitation mixture to a eukaryotic host cell lacking acell wall to form a transfection mixture with an initial Ca²⁺concentration that is at least ten fold lower than the initial Ca²⁺concentration of the precipitation mixture and wherein the precipitateis capable of remaining insoluble in the transfection mixture, and (2)diluting the precipitation mixture to form a diluted precipitationmixture, and thereafter admixing the diluted precipitation mixture to aeukaryotic host cell lacking a cell wall to form a transfection mixturewith an initial Ca²⁺ concentration that is at least ten fold lower thanthe initial Ca²⁺ concentration of the precipitation mixture and whereinthe precipitate is capable of remaining insoluble in the transfectionmixture; and incubating the transfection mixture to allow the eukaryotichost cell to take up the precipitate to form a transfected cell.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the effect of temperature and incubationtime on the DNA binding capacity of a calcium phosphate precipitateformed with 25 micrograms per milliliter (μg/ml) DNA, 125 millimoles perliter (mM) Ca²⁺ and 0.6 mM PO₄ ³⁻. Shaded, open and closed columnsrepresent incubation times of 1, 5 and 20 minutes, respectively.

FIG. 2 is a graph depicting the effect of DNA concentration andincubation time on the DNA binding capacity of a calcium phosphateprecipitate formed with 125 mM Ca²⁺ and 0.75 mM PO₄ ³⁻. Shaded, open andclosed columns represent incubation times of 1, 5 and 20 minutes,respectively.

FIG. 3 is a graph depicting the effect of Ca²⁺ concentration, incubationtemperature and incubation time on the DNA binding capacity of a calciumphosphate precipitate formed with 50 μg/ml DNA and 0.75 mM PO₄ ³⁻. TheCa²⁺ concentrations denoted as "1×", "1.2×", "1.5×", and "2×Ca"correspond to 125 mM, 150 mM, 187.5 mM and 250 mM concentrations ofCa²⁺, respectively. Shaded, open and closed columns represent incubationtimes of 1, 5 and 20 minutes, respectively.

FIG. 4 is a graph depicting the effect of DNA-calcium phosphateco-precipitation incubation time on the transient expression ofβ-galactosidase in transfected CHO or 293 host cells as determined bythe number of host cells that are positive for5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) staining as apercentage of the total population of cells. The DNA-calcium phosphateco-precipitation was carried out with 25 μg/ml DNA, 125 mM Ca²⁺, and0.75 mM PO₄ ³⁻. Shaded and closed columns represent Chinese hamstarovary (CHO) cell and human embyryonic kidney 293 cell (293 cell) hosts,respectively.

FIG. 5 is a graph depicting the effect of DNA-calcium phosphateco-precipitation incubation time on the transient expression of humantissue plasminogen activator (tPA) in transfected CHO or 293 cells asdetermined by the quantity of tPA detected in the cell culturesupernatant by ELISA, The DNA-calcium phosphate co-precipitation wascarried out with 25 μg/ml DNA, 125 mM Ca²⁺, and 0.75 mM PO₄ ³⁻. Shadedand closed columns represent CHO and 293 cell hosts, respectively.

FIG. 6 is a graph depicting the effect of DNA-calcium phosphateco-precipitation incubation time on the transient expression of tPA intransfected CHO cells as determined by the quantity of tPA detected inthe cell culture supernatant by ELISA. The DNA-calcium phosphateco-precipitation was carried out with 25 or 50 μg/ml DNA, 250 mM Ca²⁺,and 0.75 mM PO₄ ³⁻. Open and closed columns represent DNA concentrationsof 25 and 50 μg/ml, respectively.

FIG. 7 is a graph depicting the solubility of calcium phosphate as afunction of Ca²⁺ concentration and PO₄ ³⁻ concentration in a solutioncontaining 140 mM NaCl, 30 mMN-3-hydroxyethylpiperazine-N'-3-ethanesulfonic acid (HEPES), pH 7.2 at37° C.

FIG. 8 is a graph depicting the solubility of calcium phosphate as afunction of Ca²⁺ concentration and pH in a solution containing 30 mMHEPES and 0.95 mM PO₄ ³⁻ in PSO4 medium at 37° C.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

A. DEFINITIONS

As used herein, the term "transfection" is defined as the introductionof an extracellular nucleic acid into a host cell by any means known inthe art, including calcium phosphate co-precipitation, viraltransduction, liposome fusion, microinjection, microparticlebombardment, electroporation, etc. The terms "uptake of nucleic acid bya host cell", "taking up of nucleic acid by a host cell", "uptake ofparticles comprising nucleic acid by a host cell", and "taking up ofparticles comprising nucleic acid by a host cell" denote any processwherein an extracellular nucleic acid, with or without accompanyingmaterial, enters a host cell.

As used herein, the terms "nucleic acid-calcium phosphateco-precipitation" and "calcium phosphate co-precipitation" refer to aprocess wherein nucleic acid, Ca²⁺, and PO₄ ³⁻ in solution forminsoluble particles, i.e., a precipitate, comprising hydroxyapatite(which has an approximate chemical formula of (Ca₅ OH(PO₄)₃)₂, and isreferred to herein as "calcium phosphate") and nucleic acid. Alsoincluded within the definition is the growth of such particles byfurther precipitation or by aggregation and/or rearrangement of suchparticles.

As used herein, the term "calcium phosphate transfection" refers to anymethod of transfecting a host cell wherein calcium phosphate is used tofacilitate the uptake of nucleic acid by a host cell.

As used herein, the term "transformation" denotes introducing nucleicacid into a host cell so that the nucleic acid is replicable, either asa chromosomal integrant or as an extrachromosomal element.

As used herein, the term "eukaryotic host cell lacking a cell wall"refers to any nucleated cell which has no cell wall in the cell's nativestate, including all vertebrate cells, such as mammalian cells, aviancells, reptilian cells, amphibian cells, and fish cells, cells ofmulticellular invertebrate animals, such as insect cells, crustaceancells, and mollusk cells, cells of protozoans, etc., and to anynucleated cell which has had its native cell wall removed or is in anatural or artificially induced state wherein no cell wall is present,including all plant cells that are capable of forming protoplasts or arecapable of being treated to form protoplasts.

As used herein, the term "desired nucleic acid" refers to any desiredDNA, RNA or DNA/RNA hybrid.

As used herein, the term "desired DNA" is defined as anypolydeoxynucleotide, including, e.g., double stranded DNA, singlestranded DNA, double stranded DNA wherein one or both strands is (are)composed of two or more fragments, double stranded DNA wherein one orboth strands has (have) an uninterrupted phosphodiester backbone, DNAcontaining one or more single stranded portion(s) and one or more doublestranded portion(s), double stranded DNA wherein the DNA strands arefully complementary, double stranded DNA wherein the DNA strands areonly partially complementary, circular, covalently closed DNA, linearDNA, covalently cross-linked DNA, cDNA, chemically synthesized DNA,semisynthetic DNA, biosynthetic DNA, naturally isolated DNA, enzymedigested DNA, sheared DNA, plasmid DNA, chromosomal DNA, labelled DNA,such as radiolabelled DNA and fluorochrome-labelled DNA, DNA containingone or more nonnaturally occuring species of nucleic acid, etc., that isselected for transfecting a host cell.

As used herein, the term "desired RNA" is defined as anypolyribonucleotide, including, e.g., single stranded RNA, doublestranded RNA, double stranded RNA wherein one or both strands is (are)composed of two or more fragments, double stranded RNA wherein one orboth strands has (have) an uninterrupted phosphodiester backbone, RNAcontaining one or more single stranded portion(s) and one or more doublestranded portion(s), double stranded RNA wherein the RNA strands arefully complementary, double stranded RNA wherein the RNA strands areonly partially complementary, covalently cross-linked RNA, enzymedigested RNA, sheared RNA, mRNA, hnRNA, tRNA, including both charged anduncharged tRNA, rRNA, all forms of viral genomic RNA, chemicallysynthesized RNA, semisynthetic RNA, biosynthetic RNA, naturally isolatedRNA, labelled RNA, such as radiolabelled RNA and fluorochrome-labelledRNA, RNA containing one or more nonnaturally occuring species of nucleicacid, etc., that is selected for transfecting a host cell.

As used herein, the terms "desired DNA/RNA hybrid" and "desired hybridDNA/RNA" are defined as any hybrid nucleic acid comprising one strand ofDNA and one strand of RNA wherein the DNA strand and the RNA strand forma species that is at least partially double stranded, including hybridswherein the DNA strand is fully complementary or only partiallycomplementary to the RNA strand, hybrids wherein the DNA strand and/orthe RNA strand has (have) an uninterrupted phosphodiester backbone,hybrids wherein the DNA strand and/or the RNA strand is composed of twoor more fragments, hybrids containing one or more single strandedportion(s) and one or more double stranded portion(s), hybrids createdby reverse transcription of RNA, hybrids created by transcription ofDNA, hybrids created by annealing of complementary or partiallycomplementary DNA and RNA, covalently cross-linked hybrids, chemicallysynthesized hybrids, semisynthetic hybrids, biosynthetic hybrids,naturally isolated hybrids, labelled hybrids, such as radiolabelledhybrids and fluorochrome-labelled hybrids, hybrids containing one ormore nonnaturally occuring species of nucleic acid, etc.

B. GENERAL METHODS

In general, the invention provides for improved methods of calciumphosphate transfection which exploit the heretofore unknown propertiesof nucleic acid-calcium phosphate co-precipitation and calcium phosphatefacilitated nucleic acid transfection of host cells. In general, calciumphosphate transfection proceeds as follows. Nucleic acid associatesstrongly with the calcium phosphate particles formed in a calciumphosphate precipitate. In the presence of host cells, calcium phosphateparticles carrying nucleic acid precipitate onto the host cell surface,and the nucleic acid enters the host cell. The present inventorsdiscovered that there is a correlation between the host cell's abilityto take up nucleic acid and the size of the nucleic acid-calciumphosphate particles, and that there is an optimum particle size thatmaximizes the host cell's ability to take up nucleic acid. As shownherein, the present inventors have determined that the optimum particlesize is any size up to about 300 nm in length, wherein "length" isdefined as the diameter at the widest part of the particle as measuredby laser light scattering according to the method of Weiss and Frock,"Rapid Analysis of Particle Size Distribution by Laser LightScattering", Powder Technology, 14: 287 (1976). Thus, the methods of theinvention are designed to maximize the host cell's uptake of nucleicacid by exposing the host cell to particles that have an average lengthof up to about 300 nm.

It is not possible to maintain calcium phosphate particles at a constantsize because the particles are not stable in any liquid mixture. Theparticles either grow by precipitation, aggregation and rearrangement ordissolve, depending on the solubility of calcium phosphate in themixture. Accordingly, the invention provides methods wherein theparticles are grown to an optimal size in a nucleic acid-calciumphosphate co-precipitation step, the particles are diluted to lower theparticle growth rate while maintaining particle insolubility, and theoptimally sized particles are contacted with the host cell. Theinvention also provides methods wherein the particles are grown largerthan the optimal size, reduced back down to the optimal size andcontacted with the host cell.

A host cell can be exposed to the optimally-sized particles in at leastfour ways: (1) forming the optimally-sized particles in a nucleicacid-calcium phosphate co-precipitate, and then diluting theco-precipitate and contacting it with the host cell in a single step byadmixing the co-precipitate to a host cell culture; (2) forming theoptimally-sized particles in a nucleic acid-calcium phosphateco-precipitate, diluting the co-precipitate, and then admixing thediluted co-precipitate to a host cell culture; (3) forming theoptimally-sized particles in a host cell culture, and then diluting thehost cell culture; and (4) forming particles that are larger than theoptimal size, shrinking the particles back down to the optimal size andcontacting the particles with the host cells.

I. Simultaneous Dilution of Co-Precipitate and Exposure to Host Cell

a. Host Cell Preparation

Any eukaryotic host cell lacking a cell wall can be used in the methodsof the invention. Preferred for use herein are mammalian cells. Examplesof useful mammalian host cell lines include monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol., 36: 59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77: 4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23: 243-251 (1980)); monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals New York Acad. Sci., 383: 44-68 (1982)); MRC 5cells; FS4 cells; and a human hepatoma cell line (Hep G2).

The mammalian host cell of choice can be cultured by any method known inart, such as, e.g., growing the cells as a monolayer with Dulbeccomodified Eagle medium (DMEM) supplemented with 10% calf serum in anincubator at 35° C. under a 5% CO₂ atmosphere. Other procedures can beused for particular cell types. For example, Drosophila cell lines canbe grown as described by Di Nocera and Dawid, Proc. Natl. Acad. Sci.USA, 80: 7095-7098 (1983) and fish cell lines can be grown as describedby Araki et al., Bull. Natl. Res. Inst. Aquaculture, 20: 1-9 (1991).

Alternatively, a suspension cell culture can be used. Cells insuspension can be grown in spinner flasks, ranging in volume from 100milliliters (ml) to 10 liters (L) or in bioreactors ranging in volumefrom 0.5 L to 10,000 L. Cells in a suspension culture must be kept in anexponential growth phase which can be achieved by several methods knownin the art, the most common of which is subcultivation with fresh mediumevery 3 to 6 days. Standard techniques, methods and equipment arereviewed in Lubiniecki, ed, Large Scale Mammalian Cell CultureTechnology, Marcel Dekker, New York and Basle, 1990.

In the case of plant cell hosts, the plant cell protoplast culturessuitable for use herein can be prepared according to the method ofLichtenstein and Draper, "Genetic Engineering of Plants", in DNA CloningVolume III; A Practicat Approach, Glover, ed, IRL Press (1985),pp.67-119.

b. DNA Preparation

Any desired DNA for use in the methods of the invention can be preparedby a variety of methods known in the art. These methods include, but arenot limited to, chemical synthesis by any of the methods described inEngels et el., Angew. Chem. Int. Ed. Engl., 28: 716-734 (1989), theentire disclosure of which is incorporated herein by reference, such asthe triester, phosphite, phosphoramidite and H-phosphonate methods.Alternatively, the desired DNA sequences can be obtained from existingclones or, if none are available, by screening DNA libraries andconstructing the desired DNA sequences from the library clones.

Suitable quantities of DNA template for use herein can be produced byamplifying the DNA in well known cloning vectors and hosts, such asplasmid vectors carrying the pBR322 origin of replication for autonomousreplication in most Gram-negative bacterial hosts, plasmid vectorscarrying the pC194 (Ehrlich, Proc. Natl. Aced. Sci. USA. 75: 1433-1436(1978)) origin of replication for autonomous replication in Bacillus andsome other Gram-positive bacterial hosts, or 2-micron circle (2μplasmid) vectors carrying an origin of replication for autonomousreplication in most yeast hosts.

Alternatively, the DNA template can be amplified by polymerase chainreaction (PCR) as described by Seiki et el., Science, 230: 1350 (1985),Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51: 263 (1986),Mullis and Faloona, Methods Enzymol., 155: 335 (1987), and Seiki et el.,Science, 239: 487 (1988).

c. RNA Preparation

Any desired RNA for use in the methods of the invention can be preparedby a variety of methods known in the art. These methods include, but arenot limited to, chemical synthesis of RNA, and in vitro translation of aDNA template as described generally in Current Protocols in MolecularBiology, Wiley Interscience, New York (1990).

Alternatively, the desired RNA can be isolated from total cellular RNAextracted from a host cell culture. Total cellular RNA can be isolatedfrom the host cell culture by any method known in the art such as, inthe case of RNA produced in mammalian host cells, the methods describedby Favaloro et al., Methods Enzymol., 65: 718 (1980), Stallcup andWashington, J. Biol. Chem., 258: 2802 (1983), Birnboim, Nucleic AcidsRes., 16: 1487 (1988), Gilsin et al., Biochemistry, 13: 2633 (1974),Ullrich et al., Science, 196: 1313 (1977), Strohman et al., Cell, 10:265 (1977), and MacDonald et a., Methods Enzymol., 152: 219 (1987).

If the desired RNA is a polyadenylated mRNA fraction of total cellularRNA, the polyadenylated mRNA can be separated from the bulk of cellularRNA by affinity chromatography on oligodeoxythymidylate(oligo(dT))-cellulose columns using any method known in the art, such asthe method of Edmonds et al., Proc, Natl. Acad. Sci., 68: 1336 (1971) orthe method of Aviv and Leder, Proc. Acad. Sci., 69: 1408 (1972).

If the size of the desired mRNA is known, the mRNA preparation can befurther purified for mRNA molecules of the particular size by agarosegel electrophoresis of RNA in the presence of methylmercuric hydroxideas described in Lemischka et al., J. Mol. Biol., 151: 101 (1981) orfractionation of RNA by sucrose density gradient centrifugation in thepresence of methylmercuric hydroxide as described by Schweinfest et al.,Proc. Natl. Acad. Sci., 79: 4997 (1982).

In addition, the desired RNA can be obtained from the recombinant ornon-recombinant genome of an RNA virus, including single stranded RNAviruses, such as retroviruses, tobacco mosaic viruses, influenzaviruses, Newcastle disease virus, and double stranded RNA viruses suchas rotaviruses and rice dwarf virus. The desired RNA can be isolated bygrowing up the chosen RNA virus in a suitable host cell culture,harvesting the viral particles and then extracting the desired RNA fromthe viral particles. For example, the genomic RNA of Moloney's murineleukemia virus can be obtained according to the method of Schwartzberget al., Cell, 37: 1043 (1984).

d. DNA/RNA Hybrid Preparation

The DNA/RNA hybrids suitable for use in the methods of the invention canbe prepared by any method known in the art. In one embodiment, the DNAstrand or DNA fragments is (are) produced as described in Section I(b)above, the RNA strand or fragments is (are) produced as described inSection I(c) above, and the DNA and RNA strands or fragments are admixedtogether and allowed to anneal. In another embodiment, the DNA/RNAhybrid can be produced by obtaining the desired DNA strand as describedabove, using the DNA strand as a template to drive synthesis of thecomplementary RNA strand by a DNA-directed RNA polymerase, andharvesting the DNA/RNA hybrid upon completion of the transcriptionreaction. Alternatively, the DNA/RNA hybrid can be obtained by obtainingthe desired RNA strand as described above, using the RNA strand as atemplate to drive synthesis of the complementary DNA strand by aRNA-directed DNA polymerase, and harvesting the DNA/RNA hybrid uponcompletion of the reverse transcription reaction.

e. Procedure for calcium phosphate transfection

The invention encompasses any method for introducing a desired nucleicacid into a eukaryotic host cell wherein the desired nucleic acid, Ca²⁺,end PO₄ ³⁻ are admixed to form a precipitation mixture, theprecipitation mixture is incubated to form particles comprising calciumphosphate and the desired nucleic acid and the particles are allowed togrow to an average length of up to about 300 nm, the precipitationmixture is simultaneously diluted and admixed to a eukaryotic host celllacking a cell wall to form a transfection mixture wherein the particlesare capable of further growth, and the transfection mixture is incubatedto allow the host cell to take up the particles to form a transfectedcell.

1. Formation of the Precipitation Mixture Ca²⁺, PO₄ ³⁻ end the desirednucleic acid can be admixed in any order to form a precipitation mixturewherein the nucleic acid co-precipitates with calcium phosphate. In oneembodiment, the number of particles comprising nucleic acid and calciumphosphate formed in the precipitation mixture are maximized by admixingthe nucleic acid to the precipitation mixture before or simultaneouslywith the admixture of Ca²⁺ and PO₄ ³⁻. The nucleic acid can be suspendedin a buffer lacking both Ca²⁺ and PO₄ ³⁻ and then Ca²⁺ and PO₄ ³⁻ can beconsecutively or simultaneously admixed to the nucleic acid suspension.Alternatively, the nucleic acid can be suspended in a buffer containingCa²⁺ or PO₄ ³⁻ and then the appropriate counterion can be admixed to thenucleic acid suspension to initiate co-precipitation.

The concentration of reactants and the reaction conditions in theprecipitation mixture are selected to produce particles comprisingnucleic acid and calcium phosphate that have an average length of up toabout 300 nm. In a preferred embodiment, the reactant concentrations andreaction conditions are selected to produce particles that have anaverage length of about 100 nm or less. The primary factors thatdetermine particle formation are the kinetics of particle growth in themixture and the reaction time. The rate of particle growth dictates thereaction time needed to attain the desired particle size. The rate ofparticle growth is dependent on the concentration of nucleic acid andthe solubility of calcium phosphate in the mixture, and the calciumphosphate solubility, in turn, is dependent on the Ca²⁺ concentration,PO₄ ³⁻ concentration, pH, and temperature of the precipitation mixture.

The Ca²⁺ concentration, PO₄ ³⁻ concentration, pH, and temperature of theprecipitation mixture are selected to provide a calcium phosphatesolubility well below the actual Ca²⁺ concentration and PO₄ ³⁻concentration in the mixture, thus providing a supersaturation of Ca²⁺end PO₄ ³⁻ ions that drives co-precipitation of calcium phosphate andnucleic acid. Nucleic acid also affects the kinetics of particle growthin the supersaturated mixture because nucleic acid adheres to thesurface of the particles, thereby slowing particle growth.

In the precipitation mixture, Ca²⁺ can be present at an initialconcentration of about 125 mM to about 375 mM, and preferably about 250mM to about 375 mM, and PO₄ ³⁻ can be present at an initialconcentration of about 0.5 mM to about 1.0 mM and preferably about 0.75mM. The higher Ca²⁺ concentrations result in formation of particles withgreater speed and frequency. Accordingly, the optimum nucleic acidconcentration varies with the Ca²⁺ concentration in the precipitationmixture. In DNA transfection embodiments using initial Ca²⁺concentrations of about 125 mM, 250 mM and 375 mM in the precipitationmixture, the initial DNA concentrations in the precipitation mixture canbe up to about 25 μg/ml, 50 μg/ml, and 75 μg/ml, respectively. The pH ofthe precipitation mixture can be about 6.8 to about 7.6, and ispreferably about 7.05. The temperature of the precipitation mixture canbe about 0° C. to about 37° C., preferably about 20° C. to about 37° C.,and more preferably about 32° C. to about 37° C.

Any pH buffer that is effective at a pH range encompassing the desired.pH for the precipitation mixture can be used to suspend the reactants inthe precipitation mixture. Buffers that are suitable for use hereininclude appropriate concentrations ofN-3-hydroxyethylpiperazine-N'-3-ethanesulfonic acid (HEPES)-bufferedsaline, such as 25 mM HEPES and 140 mM NaCl, end appropriateconcentrations of N,N-bis(3-hydroxyethyl)-3-aminoethanesulfonic acid(BES)-buffered saline, such as 25 mM BES and 140 mM NaCl.

The precipitation mixture is incubated for a period of time sufficientto allow particles comprising calcium phosphate and nucleic acid to growto an average length of up to about 300 nm, and preferably an averagelength of about 100 nm or less. Under the reaction conditions describedabove, the particles formed in the precipitation mixture have an averagelength of about 300 nm or less after an incubation period of up to about60 seconds. Accordingly, the precipitation mixture can be incubated forup to about 60 seconds. In a preferred embodiment, the precipitationmixture is incubated for a period of about 30 seconds or less.

2. Formation of the Transfection Mixture

After particles comprising calcium phosphate and the desired nucleicacid have grown to an average length of up to about 300 nm (or less) inthe precipitation mixture, the precipitation mixture is simultaneouslydiluted and admixed to a eukaryotic host cell lacking a cell wall toform a transfection mixture. The eukaryotic cell is obtained in the formof an adherent cell culture or a suspension cell culture as described insection I(a) above. As provided herein, the precipitation mixture isdiluted by admixture to the host cell culture such that the growth rateof the particles in the transfection mixture is substantially lowered,compared to the growth rate of the particles in the precipitationmixture, without allowing resolvation of the particles, therebymaximizing the exposure of host cells to optimally sized particles.

In a preferred embodiment using a suspension cell culture, theprecipitation and dilution steps are accomplished in an automated systemwherein nucleic acid, Ca²⁺, and PO₄ ³⁻ are fed into an intake pipe thatempties into the culture vessel. The flow rate through the intake pipecan be regulated to achieve the desired incubation period for nucleicacid-calcium phosphate co-precipitation to occur within the intake pipe.Preferably, the suspension culture is agitated to maximize the contactbetween host cells and optimally sized particles of calcium phosphateand nucleic acid.

In one embodiment, the transfection mixture has an initial Ca²⁺concentration that is at least ten-fold lower than the initial Ca²⁺concentration of the precipitation mixture. In a preferred embodiment,the precipitation mixture has an initial Ca²⁺ concentration of about 250mM to about 375 mM and the transfection mixture has an initial Ca²⁺concentration of about 12 mM.

Preferably, the particle growth rate is substantially reduced by thepresence of serum or serum protein, such as bovine serum albumin, in thetransfection mixture. Protein, like nucleic acid, associates stronglywith the calcium phosphate particle surface and thereby impedes particlegrowth. In one embodiment, the transfection mixture contains about 2% toabout 10% serum, such as fetal calf serum. In another embodiment, thetransfection mixture contains about 0.2 grams per liter (g/L) to about 4g/L serum albumin, such as bovine serum albumin.

The initial PO₄ ³⁻ concentration of the transfection mixture used in thepresent methods can be conveniently provided by the PO₄ ³⁻ present inthe cell culture medium. Although other concentrations are alsoappropriate, the PO₄ ³⁻ concentration of about 1 mM inserum-supplemented cell culture media is sufficient for use herein.

The pH and temperature of the transfection mixture are maintained atphysiological levels tolerated by the host cells. In the case ofmammalian host cells, it is desirable to maintain the pH in the range ofabout 6.0 to about 8.0 and the temperature in the range of about 15° C.to about 39° C. Similarly, the transfection mixture is incubated for aperiod of time that is easily optimized for the particular host cell.

In the case of transfection in a suspension cell culture, it is possibleto precisely regulate the pH, Ca²⁺ concentration, PO₄ ³⁻ concentrationand temperature such that the solubility of the particles comprisingcalcium phosphate and the desired nucleic acid is as high as possiblewithout permitting resolvation of the particles. In a preferredembodiment, the transfection mixture is maintained at a pH of about 7.2and a temperature of about 37° C. and the Ca²⁺ concentration and the PO₄³⁻ concentration therein are maintained such that the PO₄ ³⁻ and Ca²⁺concentrations define a point that is above the calcium phosphatesolubility curve shown in FIG. 7. In another preferred embodiment, thetransfection mixture is maintained with a PO₄ ³⁻ concentration of about1 mM and a temperature of about 37° C. and the Ca²⁺ concentration and pHare maintained such that the Ca²⁺ concentration and pH define a pointabove the calcium phosphate solubility curve shown in FIG. 8.

Calcium phosphate precipitate is toxic to some host cells. Accordingly,it can be advantageous to dissolve the precipitate after the desiredincubation period for transfection. The calcium phosphate precipitate inthe transfection mixture can be dissolved, e.g., by lowering the pHand/or lowering the Ca²⁺ concentration in the transfection mixture. TheCa²⁺ concentration can be conveniently lowered by adding fresh culturemedium to the transfection mixture.

For some host cells, an improved rate of transfection is obtained byshocking the transfection mixture with glycerol or dimethylsulfoxide(DMSO) at the end of the transfection incubation period. Typically, thetransfection mixture is exposed to glycerol at a concentration of about15% volume:volume for about 30 seconds to about 3 minutes, depending onthe particular host cell, and then the cells are incubated in freshmedium for about 1 to 6 days.

Alternatively, following transfection the host cells can be cultured infresh medium for the desired time period without a glycerol shock.

II. Dilution of Co-precipitate Followed by Exposure to the Host Cell

The invention also encompasses any method for introducing a desirednucleic acid into a eukaryotic host cell wherein the desired nucleicacid, Ca²⁺, end PO₄ ³⁻ are admixed to form a precipitation mixture, theprecipitation mixture is incubated to form particles comprising calciumphosphate and the desired nucleic acid and the particles are allowed togrow to an average length of up to about 300 nm, the precipitationmixture is diluted to form a diluted precipitation mixture, the dilutedprecipitation mixture is admixed to a eukaryotic host cell lacking acell wall to form a transfection mixture wherein the particles arecapable of further growth, and the transfection mixture is incubated toallow the host cell to take up the particles to form a transfected cell.

a. Formation of the Precipitation Mixture

The precipitation mixture is obtained and incubated as described inSection I(e)(1) above. After the desired nucleic acid-calcium phosphateco-precipitate is formed, the precipitation mixture can be diluted byany convenient means, e.g., by adding an appropriate buffer or by addingthe cell culture medium to be used in transfection. Buffers and mediasuitable for use herein are described in Sections I(a) and I(e)(1)above. The diluent is added to the precipitation mixture in an amountsufficient to reduce the rate of nucleic acid-calcium phosphate particlegrowth but not allow resolvation of such particles in the resultingdiluted precipitation mixture.

Until it is admixed to a host cell to form a transfection mixture, thediluted precipitation mixture is maintained under conditions that permitcontinued but slow growth of the nucleic acid-calcium phosphateparticles. Suitable conditions for obtaining a slow particle growth rateare set forth in the description of the transfection mixture in SectionI(e)(2) above.

PATENT DOCKET 910

b. Formation of Transfection Mixture

As provided herein, the diluted precipitation mixture is admixed to aeukaryotic host cell lacking a cell wall to form a transfection mixturewherein the DNA-calcium phosphate particles will grow at a substantiallylower rate than the particle growth rate in the precipitation mixture.The eukaryotic cell is obtained in the form of an adherent cell cultureor a suspension cell culture as described in Section I(a) above, and thediluted precipitation mixture can be admixed to the cell culture to forma transfection mixture as described in Section I(e)(2) above.

The dilution of the particles in the diluted precipitation mixture endthe dilution of the particles in the transfection mixture are chosensuch that the overall dilution substantially lowers the particle growthrate without permitting the particles to dissolve. In a preferredembodiment, the overall dilution provides an initial Ca²⁺ concentrationin the transfection mixture that is at least ten-fold lower than theinitial Ca²⁺ concentration in the precipitation mixture.

In another preferred embodiment, most of the overall dilution occurs inthe formation of the diluted precipitation mixture in order to slow downparticle growth as soon as possible after the optimal particle size isattained.

Nevertheless, it will be appreciated that the invention also encompassesmethods wherein the dilution that occurs in formation of the dilutedprecipitation mixture is small in comparison to the overall dilution inthe transfection mixture. In such embodiments, it is preferable that thediluted precipitation mixture be quickly admixed to the host cellculture in order to attain a substantial decrease in particle growthrate before the average particle size exceeds the optimal range.

Alternatively, the percentage of the overall dilution that occurs in theformation of the diluted precipitation mixture and the percentage of theoverall dilution that occurs in the formation of the transfectionmixture can be varied according to the length of time between the twosteps. A short time interval would permit the use of a smaller dilutionin the diluted precipitation mixture whereas a longer time intervalwould necessitate the use of a larger dilution in the dilutedprecipitation mixture in order to prevent undue loss of transfectionactivity.

Preferably, the diluted precipitation mixture is immediately admixed tohost cells in order to maximize the host cells exposure to optimallysized nucleic acid-calcium phosphate particles. However, the inventionalso encompasses embodiments wherein the diluted precipitation mixtureis maintained for any period of time before admixture to the host cellsprovided that the diluted precipitation mixture retains some ability totransfect the host cells at the time the transfection mixture is formed.

In a preferred embodiment using a suspension cell culture, theprecipitation and dilution steps are accomplished in an automated systemwherein nucleic acid, Ca²⁺, and PO₄ ³⁻ feed into an intake pipe thatallows nucleic acid-calcium phosphate co-precipitation to occur, diluentfeeds into the precipitation mixture through another intake pipe at somepoint downstream of the nucleic acid, Ca²⁺, and PO₄ ³⁻ intake, andthereafter the diluted precipitation mixture empties into the culturevessel. The flow rate through the intake pipe that carries theprecipitation mixture and the downstream positioning of the diluentintake pipe can be adjusted to achieve the desired incubation period forthe precipitation mixture and the desired delay between dilution of theprecipitation mixture and admixture to the host cells in the culturevessel. Preferably, the suspension culture is agitated to maximize thecontact between host cells and optimally sized particles of calciumphosphate and nucleic acid.

After it is formed, the transfection mixture can be incubated under theconditions described in Section I(e)(2) above.

III. Formation of Co-Precipitate in Host Cell Culture

The invention also encompasses any method for introducing a desirednucleic acid into a eukaryotic host cell wherein Ca²⁺, PO₄ ³⁻, nucleicacid, and a eukaryotic host cell lacking a cell wall are admixed to forma precipitation mixture, the precipitation mixture is incubated to formparticles comprising calcium phosphate and the desired nucleic acid andthe particles are allowed to grow to an average length of up to about300 nm, the precipitation mixture is diluted to form a transfectionmixture wherein the particles are capable of further growth, and thetransfection mixture is incubated to allow the host cell to take up theparticles to form a transfected cell.

a. Formation of the Precipitation Mixture

A suitable host cell culture can be obtained as described in SectionI(a) above. The growth medium is removed from the cells, and the cellsare exposed to appropriate concentrations of nucleic acid, Ca²⁺, and PO₄³⁻, described in Section I(e)(1) above, to form a precipitation mixture.It will be appreciated that the order of admixing nucleic acid, Ca²⁺,and PO₄ ³⁻ is not important for practicing the invention. The cells canbe contacted with or suspended in a mixture containing any of thenucleic acid, Ca²⁺, and PO₄ ³⁻ components or combination thereof andthen admixed to any missing component or components needed to completethe precipitation mixture. Alternatively, the cells can be admixed toall of the nucleic acid, Ca²⁺, and PO₄ ³⁻ components at once.

In a preferred embodiment, the precipitation mixture is formed bycontacting the host cells with an appropriate serum-free growth mediumwhich comprises the desired concentrations of nucleic acid, Ca² +, andPO₄ ³⁻. A medium containing serum or other proteins is undesirable foruse in the precipitation mixture because proteins substantially reducethe growth of the nucleic acid-calcium phosphate co-precipitate.

The precipitation mixture reaction conditions and incubation period areselected to allow formation of optimally sized particles comprisingcalcium phosphate and nucleic acid as described in Section I(e)(1)above.

b. Formation of the Transfection Mixture

After nucleic acid-calcium phosphate particles of the desired size areformed, the precipitation mixture is diluted to form a transfectionmixture wherein the particles will grow at a substantially lower ratethan the particle growth rate in the precipitation mixture. In oneembodiment, the precipitation mixture is diluted by adding theappropriate serum-supplemented growth medium for the host cells. Theresulting transfection mixture is incubated under conditions that allowthe host cell to take up the nucleic acid-calcium phosphate particles toform a transfected cell. Such procedures are described in SectionI(e)(2) above.

IV. Shrinking an Overgrown Co-Precipitate Followed by Exposure to theHost Cell

The invention further encompasses any method for introducing a desirednucleic acid into a eukaryotic host cell wherein Ca²⁺, PO₄ ³⁻ andnucleic acid are admixed to form a precipitation mixture, theprecipitation mixture is incubated to form particles comprising calciumphosphate and the desired nucleic acid and the particles are allowed togrow to an average length that is greater than about 300 nm, theparticles are decreased in size to form optimally-sized particles withan average length of about 300 nm or less by incubating theprecipitation mixture under conditions wherein the particles are capableof resolvation, the optimally-sized particles are admixed to aeukaryotic host cell lacking a cell wall to form a transfection mixture,and the transfection mixture is incubated to allow the host cell to takeup the particles to form a transfected cell.

a. Formation of the Overgrown Precipitate

An overgrown nucleic acid-calcium phosphate co-precipitate can beobtained by creating a precipitation mixture as described in SectionI(e)(1) above and incubating the precipitation mixture under conditionsthat allow particles comprising calcium phosphate and nucleic acid togrow to an average length that is greater than about 300 nm. Under thereaction conditions described in Section I(e)(1) above, the particlesformed in the precipitation mixture have an average length of about 300nm or less after an incubation period of about 60 seconds. Accordingly,an overgrown precipitate can be formed by incubating the precipitationmixture for at least about 60 seconds.

b. Shrinking the Overgrown Precipitate

After the overgrown precipitate is formed in the precipitation mixture,the conditions in the precipitation mixture are altered to causereduction of particle bulk in the precipitate. Since particle growth orshrinkage is dependent upon the solubility of calcium phosphate in theprecipitation mixture, particle shrinkage can be achieved by increasingthe solubility of calcium phosphate in the precipitation mixture. Asdescribed above, calcium phosphate solubility is determined by Ca²⁺concentration, PO₄ ³⁻ concentration, pH and temperature. Calciumphosphate solubility can be increased by lowering the Ca²⁺concentration, lowering the PO₄ ³⁻ concentration, lowering the pH and/orlowering the temperature of the precipitation mixture. In a preferredembodiment, the precipitation mixture is diluted with an appropriatebuffer to lower the Ca²⁺ and PO₄ ³⁻ concentrations. In anotherembodiment, the dilution is performed by centrifuging the precipitationmixture and resuspending the pellet in an appropriate buffer to createan undersaturated solution of Ca²⁺ and PO₄ ³⁻. In yet another preferredembodiment, the pH of the precipitation mixture is lowered to create anundersaturated solution of Ca²⁺ and PO₄ ³⁻.

The precipitation mixture is incubated under conditions permittingshrinkage of the particles until the particles reach the optimal averagelength of about 300 nm or less, and preferably about 100 nm or less. Theincubation period necessary to form optimally-sized particles variesaccording to the size of the overgrown particles immediately prior toshrinkage and the speed of resolvation in the precipitation mixture.Thus, the incubation time is selected based on the amount of particlesize reduction needed and the rate of particle shrinkage set by the Ca²⁺and PO₄ ³⁻ undersaturation in the precipitation mixture.

c. Formation of Transfection Mixture

After the nucleic acid-calcium phosphate particles in the precipitationmixture are reduced to the optimal size, the conditions in theprecipitation mixture are changed again to permit slow growth of theoptimally-sized particles. In one embodiment, the precipitation mixtureis admixed to a eukaryotic host cell lacking a cell wall to form atransfection mixture with an adequate Ca²⁺ concentration, PO₄ ³⁻concentration, pH and temperature to permit the particles to grow at aslow rate as described in Section I(e)(2) above. In another embodiment,the Ca²⁺ concentration, PO₄ ³⁻ concentration, pH and temperature of theprecipitation mixture, or any one or combination of these parameters, is(are) increased to attain a Ca²⁺ and PO₄ ³⁻ supersaturation level thatpermits the particles to grow at a slow rate and the precipitationmixture is admixed to a eukaryotic host cell lacking a cell wall to forma transfection mixture.

Lastly, the transfection mixture is incubated as described in SectionI(e)(2) above.

V. Other Embodiments Involving Shrinkage of an Overgrown Co-Precipitate

In addition to methods for shrinking an overgrown precipitate prior tocontacting the precipitate and the host cell, the invention alsoencompasses methods wherein the overgrown nucleic acid-calcium phosphateparticles are simultaneously reduced in size and exposed to the hostcell. In these embodiments, the precipitation mixture is simultaneouslydiluted and admixed to a host cell culture to form a transfectionmixture wherein the particles are capable of decreasing in size and thetransfection mixture is incubated under conditions wherein the particlesare reduced until they reach an average length of about 300 nm or less,and preferably about 100 nm or less. The rate of particle resolvation(determined by the Ca²⁺ concentration, PO₄ ³⁻ concentration, pH, andtemperature of the transfection mixture) and incubation time areselected to provide the desired amount of particle reduction asdescribed in Section IV(b) above. Next, the conditions in thetransfection mixture are changed to permit slow growth of the particlesand the transfection mixture is incubated to allow the host cell to takeup the particles to form a transformed host cell as described in SectionIV(c) above.

Alternatively, an overgrown precipitate can be simultaneously dilutedand admixed to a host cell culture to form a transfection mixturewherein the particles slowly decrease in size until they completelydissolve, which allows the host cell to take up the optimally sizedparticles that exist when the particles are reduced to an average lengthof 300 nm or less. In a preferred embodiment, the reduction in particlesize is effected by allowing the transfection mixture to slowly lowerits pH as a result of the CO₂ and lactate production of the host cellsin culture. The gradual decrease in pH causes the transfection mixtureto change from oversaturation of Ca²⁺ and PO₄ ³⁻ to an undersaturationof Ca²⁺ end PO₄ ³⁻, end the resulting slow rate of particle sizereduction maximizes the time period during which host cells are exposedto optimally sized particles.

In another embodiment, the initial resolvation of overgrown particles inthe host cell culture is followed by repeated cycles of admixing freshovergrown precipitate to the transfection mixture and dissolving theprecipitate in the transfection mixture until the desired level oftransfection is obtained.

Also encompassed herein are methods wherein the co-precipitation occursin the host cell culture (as described in Section III above) and isallowed to continue until the particles are larger than the optimalsize. In these embodiments, the overgrown particles are reduced backdown to the optimal size by changing the culture conditions, and afteroptimal particle size is reached the culture conditions are changedagain to permit slow growth of the particles and allow the host cell totake up the particles to form a transfected cell.

In addition, all of the methods described herein can be modified toincorporate a continuous cycle of particle overgrowth/particle reductionduring incubation of the transfection mixture designed to maximize theamount of time that the host cells are exposed to optimally-sizedparticles. In a preferred embodiment using a suspension culture in acomputer-controlled bioreactor, conditions in the transfection mixtureare automatically altered according to an algorithm that estimatesparticle size and calculates the timing and degree of condition changesneeded to achieve maximum cell contact with optimally-sized particles.

Further details of the invention can be found in the following examples,which further define the scope of the invention. All references citedherein are expressly incorporated by reference in their entirety.

EXAMPLE 1 Materials and Methods

1. Precipitation

Various amounts of purified plasmid DNA of the β-galactosidaseexpression vector pSVβ (Clontech, Palo Alto, Calif.) or of the tPAexpression vector described in Refino et al., Thrombosis and Hematosis,70: 313-319 (1993) were diluted in a TE buffer (1 mMTris(hydroxymethyl)aminomethane (Tris), 0.1 mMethylenediamine-tetraacetic acid (EDTA), pH 7.6) solution containingconcentrations of CaCl₂ that varied from 250 mM to 500 mM. One volume ofthe DNA/CaCl₂ solution was quickly added to one volume of a phosphatesolution (50 mM HEPES, 280 mM NaCl, 1.5 mM Na₂ HPO₄, pH 7.05) and themixture was gently vortex mixed to initiate the formation of theprecipitate. After various time periods of incubation at varioustemperatures, 300 μl aliquots of the mixture were microcentrifuged for45 seconds and the optical densities of the supematants were measured at260 nm (OD₂₆₀) to quantify the amount of unbound DNA (an OD₂₆₀ of 1.0corresponded to a DNA concentration of 50 μg/μl).

2. Transfection

CHO cells (CHO-DUKX DHFR minus, Urlaub and Chasin, Proc. Natl. Acad.Sci. USA, 77: 4216-4220 (1980)) and human kidney 293 cells (ATCCCRL1573) for transfactions were passaged regularly every 3 to 4 days bysubcultivation in T-flasks or in 500 ml spinner flasks using PSO4medium, a medium proprietary to Genentech, Inc. (developed from theoriginal formulations of Dulbecco's modified Eagle medium and Ham's F12medium, catalog numbers 430-3000EB and 430-1700EB in the 1990 catalog ofGibco BRL, Gaithersburg, Md. 20877), containing 2% to 10% fetal calfserum. Cell cultures were conducted according to the general methodsdescribed in Tissue Culture: Laboratory Procedures, Doyle, Griffiths andNewell, ads, J. Wiley and Sons, New York (1992). One day prior totransfection, cells taken from exponentially growing cultures wereseeded into 12 well plates at a density of 2×10⁵ cells per well, 1milliliter (ml) of growth medium supplemented with 2% to 10% fetal calfserum was added to each well, and the cell culture plates were incubatedat 37° C. under a 5% CO₂ atmosphere for 24 hours.

The precipitation mixture was added to the individual wells (aftervarious time periods of incubation at various temperatures) involume:volume (v/v) ratios that resulted in a final Ca²⁺ concentrationof 12.5 mM. This maintained the condition of Ca²⁺ and PO₄ ³⁻supersaturation if the pH was held at or above 7.2. CHO cells wereexposed to the precipitate for 3 to 6 hours, shocked for 30 seconds with15% glycerol, washed once and then incubated in fresh growth medium for1 to 6 days. 293 cells were exposed to the precipitate for 3 to 20 hoursand then incubated in fresh growth medium, without a glycerol shock, for1 to 6 days.

Results and Discussion

A. Physico-chemical parameters of calcium phosphate DNA co-precipitate

1. Temperature

Although the heretofore disclosed calcium phosphate transfectionprotocols provide that DNA-calcium phosphate co-precipitation be carriedout for at least 10 minutes, and most protocols recommend 20 minutes ofco-precipitation, it was determined that under standard conditions (125mM Ca² +, 0.75 mM PO₄ ³⁻, 25 μg/ml DNA, 20° C., pH 7.05) more than 95%of the DNA bound to the precipitate in less than 1 minute afteradmixture of the DNA/CaCl₂ solution to the phosphate solution (see thedata for 25 μg/ml DNA in FIG. 2). To facilitate an investigation of theeffect of temperature, the rate of crystal growth was reduced by using alower phosphate concentration (0.6 mM PO₄ ³⁻). The reduced rate ofcrystal growth permitted the binding of DNA to the precipitate to beobserved over a period of 20 minutes at temperatures between 0° C. and37° C. (FIG. 1). At 37° C., 100% of the DNA bound to the precipitatewithin 1 minute whereas at 0° C., all of the DNA remained in solutionfor 20 minutes. This result is due to the higher solubility of calciumphosphate at lower temperatures, which yields a less oversaturatedsolution with a lowered frequency of crystal nucleation.

2. DNA concentration

Since DNA concentration affects the transfection efficiency, theinfluence of DNA concentration on DNA-calcium phosphate co-precipitationwas determined (FIG. 2). The precipitation was carried out at 20° C.with a phosphate concentration of 0.75 mM. The results demonstrated thatan increase in the DNA concentration from 25 μg/ml to 50 μg/mldramatically reduced the binding capacity of the precipitate (FIG. 2).At a concentration of 50 μg/ml, over 90% of the DNA remained in solutionafter 20 minutes and almost no pellet was found upon microcentrifugationfor 45 seconds.

3. Calcium concentration

Although Ca²⁺ was present at a concentration that was 170-fold greaterthan the concentration of PO₄ ³⁺ in the precipitation mixture, a furtherincrease in Ca²⁺ strongly affected the formation of a precipitate (FIG.3). In the presence of 250 mM Ca²⁺ and 50 μg/ml DNA, 30 to 45 μg/ml DNAwas bound to a precipitate. Given that the absolute amount ofprecipitate was limited by the phosphate concentration (0.75 mM), amaximum of 125 μg of precipitate per ml was expected if all phosphateprecipitated. The precipitate was found to bind up to 50 μg/ml of DNA inthe presence of 375 mM Ca²⁺ and 75 μg/ml DNA. Thus, DNA can account forapproximately 40% of the mass of a precipitate that is formed underoptimal conditions.

B. Transient Expression in CHO and 293 Cells

The DNA-calcium phosphate co-precipitation was carried out understandard conditions (25 μg/ml DNA, 125 mM Ca²⁺, 0.75 PO₄ ³⁻). Afterincubation at 20° C. for various time periods, the precipitate wasadmixed to an exponentially growing cell culture. The transfectionefficiency was analyzed either in an intracellular β-galactosidaseexpression system wherein expressing cells were identified by X-galstaining according to the method of Somes et al., EMBO, 5: 3133-3142(1986) or in a tPA expression system wherein. secreted tPA product wasquantified with an ELISA assay according to the method of Bennet et al.,J. Biol. Chem., 266: 5191-5201 (1991). As shown in FIGS. 4 and 5, a oneminute precipitation step produced the highest DNA transfectionefficiency and expression level among the precipitation incubationperiods tested. Inspection of the precipitates under a light microscope(phase contrast, 20× magnification) after 4 hours of exposure to thehost cells showed that 1 minute of precipitation resulted in theformation of an enormous number of very small particles whereas 20minutes of precipitation resulted in the formation of bigger but muchfewer particles. The high transfection efficiency of the one-minuteprecipitates in both 293 cells and CHO cells is consistent with theabove-described data showing that 1 minute of precipitation wassufficient to bind 100% of the DNA in a precipitation mixture with 25μg/ml DNA, 125 mM Ca²⁺ and 0.75 mM PO₄ ³.

High levels of transient expression were achieved by using a Ca²⁺concentration of 250 mM (which maximized the frequency of the crystalnucleation event) and a DNA concentration of 50 μg/ml (which saturatedthe growing crystals). The crystal size was controlled by admixing theprecipitate to the cells after very short incubation periods as shown inFIG. 6. Under these conditions, tPA product titers obtained in CHO hostcells were as high as 2 μg/ml and were routinely above 1 μg/ml, 3 daysafter transfection. 293 cells also produced higher transient expressionlevels after transfection with precipitates formed in 30 seconds with250 mM Ca²⁺ and 50 μg/ml DNA. Under these conditions, 293 cells yielded5 μ/ml of tPA 2 days after transfection with a transient specificproductivity of about 2 μg/10⁶ cells/day.

EXAMPLE 2 Materials and Methods

In a first matrix plate test, various concentrations of PO₄ ³⁻ (between0.4 mM end 2.5 mM) were combined with various concentrations of Ca²⁺(between 2 mM end 10 mM) in 1 ml of 140 mM NaCl, 30 mM HEPES, pH 7.2.The plates were incubated overnight at 37° C. Each well was examinedunder a light microscope (phase contrast, 20× magnification) and scoredfor the presence of a precipitate.

In a second matrix plate test, various concentrations of Ca²⁺ (between4.2 mM and 18.0 mM) were combined with 1 ml of 30 mM HEPES and 0.95 mMPO₄ ³⁻ in PSO4 medium at a various pH's (between 6.8 and 7.6). Themixtures were incubated overnight at 37° C. Each well was examined undera light microscope (phase contrast, 20× magnification) and scored forthe presence of a precipitate.

Results and Discussion

The data produced in the first set of tests are summarized in FIG. 7.The data points on the curve in FIG. 7 represent the lowestconcentrations of Ca²⁺ and PO₄ ³⁻ that produced a precipitate. Thus, thedata points appearing in FIG. 7 define a calcium phosphate solubilitycurve as a function of Ca²⁺ concentration and PO₄ ³⁻ concentration.Using a regression with the method of least squares, the solubility ofcalcium phosphate as a function of Ca²⁺ concentration and PO₄ ³⁻concentration was found to be approximated by the equationy=(4.704)(x⁻⁰.82699), where y is the Ca²⁺ concentration and x is the PO₄³⁻ concentration. At any point (x,y) above the curve, the calciumphosphate particles are capable of continued growth. At any point (x,y)below the curve, the precipitate quickly redissolves.

The data produced in the second set of tests are summarized in FIG. 8.The data points on the curve in FIG. 8 represent the lowest Ca²⁺concentrations and pH's that produced a precipitate. Thus, the datapoints appearing in FIG. 8 define a calcium phosphate solubility curveas a function of Ca²⁺ concentration and pH. Using a regression with themethod of least squares, the solubility of calcium phosphate as afunction of Ca²⁺ concentration and pH was found to be approximated bythe equation y=(8.6886)(10¹²)(x⁻¹⁴.075), where y is the Ca²⁺concentration and x is the pH. At any point (x,y) above the curve, thecalcium phosphate particles are capable of continued growth. At anypoint (x,y) below the curve, the precipitate quickly redissolves.

We claim:
 1. A method for introducing a desired nucleic acid into aeukeryotic host cell, comprising(a) admixing Ca²⁺, PO₄ ³⁻ and thedesired nucleic acid to form a precipitation mixture; (b) incubating theprecipitation mixture to form particles comprising calcium phosphate andthe desired nucleic acid, and allowing the particles to grow to anaverage length of up to about 300 nm; (c) performing a step selectedfrom the group consisting of: (1) diluting the precipitation mixture andsimultaneously admixing the precipitation mixture with a eukaryotic hostcell lacking a cell wall to form a transfection mixture; and (2)diluting the precipitation mixture to form a diluted precipitationmixture, and thereafter admixing the diluted precipitation mixture witha eukaryotic host cell lacking a cell wall to form a transfectionmixture; and (d) incubating the transfection mixture to allow theeukaryoric host cell to take up the particles to form a transfectedcell.
 2. The method of claim 1 wherein in step (b) the particles areallowed to grow to an average length of up to about 100 nm.
 3. Themethod of claim 1 wherein the eukaryotic host cell is a mammalian cell.4. The method of claim 1 wherein in step (b) the precipitation mixtureis incubated for a period of up to about 60 seconds.
 5. The method ofclaim 4 wherein in step (b) the precipitation mixture is incubated for aperiod of up to about 30 seconds.
 6. The method of claim 1 wherein thedesired nucleic acid is DNA.
 7. The method of claim 6 wherein in step(a) the precipitation mixture comprises an initial Ca²⁺ concentration ofabout 250 mM.
 8. The method of claim 7 wherein in step (a) theprecipitation mixture comprises an initial concentration of the desiredDNA of about 50 μg/ml.
 9. The method of claim 6 wherein in step (a) theprecipitation mixture comprises an initial Ca²⁺ concentration of about375 mM and an initial concentration of the desired DNA of about 75μg/ml.
 10. The method of claim 1 wherein in step (d) the transfectionmixture comprises a pH of about 7.2, a Ca²⁺ concentration equal to avalue y expressed as millimoles per liter (mM), end a PO₄ ³⁻concentration equal to a value x expressed as mM, wherein y is about 2.0to about 20.0, x is about 0.4 to about 2.5, and y is greater than(4.704)(x⁻⁰.82669).
 11. The method of claim 1 wherein in step (d) thetransfection mixture comprises a PO₄ ³⁻ concentration of about 1.0 mM, aCa²⁺ concentration equal to a value y expressed as millimoles per liter(mM) and a pH equal to a value x expressed as -log₁₀ (moles of H⁺ perliter), wherein y is about 3.0 to about 20.0, x is about 6.8 to about7.8, and y is greater than (8.6886) (10¹²)(x⁻¹⁴.075).
 12. The method ofclaim 1 wherein in step (c) the transfection mixture contains an initialCa²⁺ concentration that is at least about ten fold lower than theinitial Ca²⁺ concentration of the precipitation mixture in step (b).